The mu opioid receptor, the type most closely associated with both analgesia and behavioral reinforcement, is subject to a variety of factors that regulate its effectiveness in eliciting a cellular response. Among these are the structure of the receptor itself, its density in the cell membrane, and its proximity to coupling proteins. In addition, these factors are all linked by dynamic interactions whose timing, only partially understood at present, can have important influence on the performance of a drug. During the past year we have continued investigations into these factors and dynamics in two ways. The first employed confocal microscopy and a high affinity fluorescent antagonist, "FNAL". The second is the ongoing study of the irreversible antagonist beta-FNA to determine whether a mutant receptor, H297Q, remains capable of covalent modification by the drug and how its response to the drug differs from that of the wild-type receptor. The results of the two programs follow: (1) Confocal microscopy is not only useful to localize probes at a subcellular level, but it can also quantitate them on living cells in real time, as we first showed in 2000. The method is unique in its ability to optically section biological material, and we employ this capability to demonstrate binding kinetics that depend on the vertical position of a cell's membrane. This year, using the accumulated perfections of the previous year, we have concluded the experimental program that was set out by Richard Shrager, who has also analyzed all the results. (2) The kinetic studies with beta-FNA have continued this year using a higher level of precision. In particular, we found that the rate constants derived from our first studies were prone to large errors due to errors in estimating the receptor concentration. We devised a method for estimating the latter that could give < 10% error using only 4 duplicate data points. Using this assay method, we found that by controlling the number of days cells mature in culture as well as by pre-treatment of the cells with the antagonist naloxone, we could overcome a large difficulty of the earlier work, namely that of matching receptor concentrations in cells expressing wild-type and mutant receptors. Having now surmounted these obstacles, we have concluded the kinetic studies. Along the way we encountered new questions regarding the physiological regulation of mu opioid receptor. Among them are (1) the two-fold disparity in receptor density as measured by labeled naloxone compared to labeled beta-FNA when (and only when) living cells are disrupted, (2) the sensitivity of "nonspecific" binding of naloxone to nutrients in the binding medium, and (3) the nature of the displacement of naloxone by drugs as opposed to its dissociation by simple dilution. These questions are now the subjects of our current investigations.